Method of treating hepatitis virus infection with a multiphasic interferon delivery profile

ABSTRACT

The present invention provides methods of treating hepatitis virus infection. The methods generally involve administering a composition comprising an antiviral agent in a dosing regimen that achieves a multiphasic serum concentration profile of the antiviral agent. The dosing regiment includes dosing events that are less frequent than with currently available hepatitis therapies. The multiphasic antiviral agent serum concentration profile that is achieved using the methods of the invention effects an initial rapid drop in viral titer, followed by a further decrease in viral titer over time, to achieve a sustained viral response

FIELD OF THE INVENTION

This invention is in the field of treatments for viral infections, inparticular hepatitis virus.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is the most common chronic blood borneinfection in the United States. Although the numbers of new infectionshave declined, the burden of chronic infection is substantial, withCenters for Disease Control estimates of 3.9 million (1.8%) infectedpersons in the United States. Chronic liver disease is the tenth leadingcause of death among adults in the United States, and accounts forapproximately 25,000 deaths annually, or approximately 1% of all deaths.Studies indicate that 40% of chronic liver disease is HCV-related,resulting in an estimated 8,000-10,000 deaths each year. HCV-associatedend-stage liver disease is the most frequent indication for livertransplantation among adults.

Antiviral therapy of chronic hepatitis C has evolved rapidly over thelast decade, with significant improvements seen in the efficacy oftreatment. Nevertheless, even with combination therapy using pegylatedIFN-α plus ribavirin, 40% to 50% of patients fail therapy, i.e., arenonresponders or relapsers. These patients currently have no effectivetherapeutic alternative. In particular, patients who have advancedfibrosis or cirrhosis on liver biopsy are at significant risk ofdeveloping complications of advanced liver disease, including ascites,jaundice, variceal bleeding, encephalopathy, and progressive liverfailure, as well as a markedly increased risk of hepatocellularcarcinoma.

The high prevalence of chronic HCV infection has important public healthimplications for the future burden of chronic liver disease in theUnited States. Data derived from the National Health and NutritionExamination Survey (NHANES III) indicate that a large increase in therate of new HCV infections occurred from the late 1960s to the early1980s, particularly among persons between 20 to 40 years of age. It isestimated that the number of persons with long-standing HCV infection of20 years or longer could more than quadruple from 1990 to 2015, from750,000 to over 3 million. The proportional increase in persons infectedfor 30 or 40 years would be even greater. Since the risk of HCV-relatedchronic liver disease is related to the duration of infection, with therisk of cirrhosis progressively increasing for persons infected forlonger than 20 years, this will result in a substantial increase incirrhosis-related morbidity and mortality among patients infectedbetween the years of 1965-1985.

Chronic hepatitis C virus infection is characterized by intermittent orpersistent elevations in serum alanine aminotransferase (ALT) levels andconstant levels of HCV RNA in the circulation. Currently, approvedtherapies use alpha interferons derived from natural leukocytes or byrecombinant methods using cDNA sequences of specific subtypes orconsensus interferon-α (IFN-α). The accepted dosage regimen is asubcutaneous administration of IFN-α in the dose ranges of 6-50 μg threetimes in week for a period of 24-48 weeks.

Cyclical administration of IFN-α has also been conducted, in the hopethat viral clearance can be achieved. The repeat dosing has been deemednecessary in view of the rapid clearance and in vivo degradation ofIFN-α. In another attempt to achieve better efficacy, combinationtherapies such as IFN-α and ribavirin have been carried out. In patientsinfected with the genotype 1 virus, which is the most prevalent HCVstrain, only ≦25% of the patients demonstrated sustained viral responseeven with combination therapy. In attempts to improve further thetherapeutic methods, various investigators have attempted a chemicalmodification of IFN-α by adding a polymer chain(s) to increase themolecular weight and size of the protein and to prolong the systemiccirculation times. While these manipulations of IFN-α increased thecirculation times and improved the efficacies further, a significantfraction of the protein loses its biological activity. Thus higheramounts of the protein have to be delivered to the patient with adverseeffects such as neutropenia accompanying such administrations.

Viral kinetics during treatment regimens that include IFN-α have beenexamined. In general, an initial rapid decline in viral titers (earlyviral response; EVR) is seen in some individuals. The EVR results in anapproximately 0.5- to 3-log decrease in serum HCV RNA levels in a periodof 24-48 hours after initiation of treatment. An early robust responseis favorable toward achieving a durable response. In some individuals,the EVR is followed by a further, less rapid decline of the virus inblood (second phase decline). The second phase decline is a slowerdecrease in the level of the virus over several weeks or months.

Despite the availability of approved treatment regimens discussed above,only a small fraction of the individuals treated attain a sustainedviral response. Thus, there is a need in the art for improved methodsfor treating HCV infection. The present invention addresses this need.

Literature

U.S. Pat. Nos. 6,172,046; 6,245,740; 5,824,784; 5,372,808; 5,980,884;published international patent applications WO 96/21468; WO 96/11953;Torre et al. (2001) J. Med. Virol. 64:455-459; Bekkering et al. (2001)J. Hepatol. 34:435440; Zeuzem et al. (2001) Gastroenterol.120:1438-1447; Zeuzem (1999) J. Hepatol. 31:61-64; Keeffe and Hollinger(1997) Hepatol. 26:101s-107S; Wills (1990) Clin. Pharmacokinet.19:390-399; Heathcote et al. (2000) New Engl. J. Med. 343:1673-1680;Husa and Husova (2001) Bratisl. Lek. Listy 102:248-252; Glue et al.(2000) Clin. Pharmacol. 68:556-567; Bailon et al. (2001) Bioconj. Chem.12:195-202; and Neumann et al. (2001) Science 282:103; Zalipsky (1995)Adv. Drug Delivery Reviews S. 16, 157-182; Mann et al. (2001) Lancet358:958-965.

SUMMARY OF THE INVENTION

The present invention provides methods of treating hepatitis virusinfection. The methods generally involve administering a compositioncomprising an antiviral agent in a dosing regimen that achieves amultiphasic serum concentration profile of the antiviral agent. Thedosing regimen includes dosing events that are less frequent than withcurrently available hepatitis therapies. The multiphasic antiviral agentserum concentration profile that is achieved using the methods of theinvention effects an initial rapid drop in viral titer, followed by afurther decrease in viral titer over time, to achieve a sustained viralresponse.

FEATURES OF THE INVENTION

In some embodiments, the invention features a method for treatinghepatitis C virus infection in an individual. The method generallyinvolves administering a composition comprising interferon-α (IFN-α) inan amount effective to achieve a first serum concentration of IFN-α thatis at least about 80% of the maximum tolerated dose (MTD) within a firstperiod of time of about 24 to 48 hours, followed by a secondconcentration of IFN-α that is about 50% or less than the MTD, whichsecond concentration is maintained for a second period of time of atleast seven days. In some embodiments, a sustained viral response isachieved.

In some embodiments, the methods further include administering IFN-γ fora period of from about 1 day to about 14 days before administration ofIFN-α.

In some embodiments, lFN-α is administered in a depot. In otherembodiments, IFN-α is administered by continuous infusion. In someembodiments, continuous infusion administration is achieved with a pump.In other embodiments, IFN-α is administered by a single subcutaneousinjection followed by continuous infusion using a pump.

In some embodiments, the invention features a method of treatinghepatitis C virus infection in an individual, the method generallyinvolving administering IFN-α in a dosing regimen comprising a firstphase and a second phase, wherein, in the first phase, a first serumconcentration of IFN-α is achieved that is at least about 80% of themaximum tolerated dose (MTD) within a first period of time of about 24hours, wherein in the second phase, the ratio of the highest IFN-α serumconcentration to the lowest serum IFN-α concentration, measured over any24-hour period during the second phase, is less than 3, and wherein thehighest concentration of IFN-α during the second phase is about 50% orless than the MTD. In some of these embodiments, the ratio of thehighest IFN-α serum concentration to the lowest serum IFN-αconcentration, measured over any 24-hour period during the second periodof time is about 1.

In some embodiments, the invention features a method of treatinghepatitis C virus infection in an individual, the method generallyinvolving administering a composition comprising consensus interferon-α(CIFN) in an amount effective to achieve a first serum concentration ofCIFN that is at least about 80% of the maximum tolerated dose (MTD)within a first period of time of about 24 hours, followed by a secondconcentration of CIFN that is about 50% or less than the MTD, whichsecond concentration is maintained for a second period of time of atleast seven days.

In some embodiments, the invention features a method of treatinghepatitis C virus infection in an individual, the method generallyinvolving administering consensus IFN-α (CIFN) in a dosing regimencomprising a first phase and a second phase, wherein, in the firstphase, a first serum concentration of CIFN is achieved that is at leastabout 80% of the maximum tolerated dose (MTD) within a first period oftime of about 24 hours, wherein in the second phase, the ratio of thehighest CIFN serum concentration to the lowest serum CIFN concentration,measured over any 24-hour period during the second phase, is less than3, and wherein the highest concentration of CIFN during the second phaseis about 50% or less than the MTD.

In some embodiments, the invention features a method of treatinghepatitis C virus infection in an individual, the method generallyinvolving administering IFN-α in a dosing regimen comprising a firstphase and a second phase, wherein, in the first phase, a first serumconcentration C1max of IFN-α is achieved within a first period of timeof about 24 hours, wherein in the second phase, a Csus is achieved thatis about 50% of C1max or less, and wherein the area under the curve,defined by IFN-α serum concentration as a function of time, during any24-hour time period in the second phase is no greater than the areaunder the curve of day 2 to day 3 as shown in FIG. 2.

In some embodiments, the invention features a method of treatinghepatitis C virus infection in an individual, the method generallyinvolving administering consensus IFN-α (CIFN) in a dosing regimencomprising a first phase and a second phase, wherein, in the firstphase, a first serum concentration C1max of CIFN is achieved within afirst period of time of about 24 hours, wherein in the second phase, aCsus is achieved that is about 50% of C1max or less, and wherein thearea under the curve, defined by CIFN serum concentration as a functionof time, during any 24-hour time period in the second phase is nogreater than the area under the curve of day 2 to day 3 as shown in FIG.2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting a viral kinetics during interferon-α therapydepicted here as clearance of HCV virus in blood as monitored by thelevel of viral RNA in serum using a sensitive measurement such as apolymerase chain reaction.

FIG. 2 is a graph depicting a profile of serum IFN-α concentrationduring administration of a controlled Release Injectible (CRI) system ora zero-order throughput system and bolus. Viral kinetics followingconventional TIW regimen is included to contrast the improvements with atherapeutic dosing regimen according to the instant invention (seebelow). FIG. 3 is a graph depicting a profile of serum IFN-αconcentration during administration of a controlled release injectible(CRI). In one scenario, the early phase concentration improves theinitial viral decline (see dotted line).

FIG. 4 is a graph depicting the viral kinetics and pharmacokineticsfollowing a CRI therapy. In this scenario, the early viral response(EVR) is similar to conventional TIW therapy. The high concentrationCsus in second phase affects the slope, making the slope steep (seedotted line).

FIG. 5 is a graph depicting the viral kinetics and pharmacokineticsfollowing a CRI therapy using IFN-α. In this scenario, there issignificantly more decline in early viral titers and the second phasealso exhibits a steep decline (see dotted line).

FIG. 6 is a graph depicting viral kinetics during administration ofIFN-α with a sustained release delivery system providing repeat Cmax andCsus concentrations of drug to achieve significant sustained viralresponse. Because of repeat Cmaxs and sustained high Csus, a drop inviral titer can be seen as step (see dotted line).

DEFINITIONS

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease (as in liver fibrosisthat can result in the context of chronic HCV infection); (b) inhibitingthe disease, i.e., arresting its development; and (c) relieving thedisease, i.e., causing regression of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, primates, including simians and humans.

The term “early viral response (EVR),” used interchangeably with“initial viral response,” “rapid viral response” refers to the drop inviral titer within about 24 hours, about 48 hours, about 3 days, orabout 1 week after the beginning of treatment for HCV infection.

The term “second phase decline” as used herein refers to a slowerdecrease in the level of the virus over several weeks or months afterthe EVR.

The term “sustained viral response” (SVR; also referred to as a“sustained response” or a “durable response”), as used herein, refers tothe response of an individual to a treatment regimen for HCV infection,in terms of serum HCV titer. Generally, a “sustained viral response”refers to no detectable HCV RNA (e.g., less than about 500, less thanabout 200, or less than about 100 genome copies per milliliter serum)found in the patient's serum for a period of at least about one month,at least about two months, at least about three months, at least aboutfour months, at least about five months, or at least about six monthsfollowing cessation of treatment.

“Treatment failure patients” as used herein generally refers toHCV-infected patients who failed to respond to previous therapy for HCV(referred to as “non-responders”) or who initially responded to previoustherapy, but in whom the therapeutic response was not maintained(referred to as “relapsers”). The previous therapy generally can includetreatment with IFN-α monotherapy or IFN-α combination therapy, where thecombination therapy may include administration of IFN-α and an antiviralagent such as ribavirin.

The term “hepatitis virus infection” refers to infection with one ormore of hepatitis A, B, C, D, or E virus, with blood-borne hepatitisviral infection being of particular interest.

As used herein, the term “hepatic fibrosis,” used interchangeably hereinwith “liver fibrosis,” refers to the growth of scar tissue in the liverthat can occur in the context of a chronic hepatitis infection.

As used herein, the term “liver function” refers to a normal function ofthe liver, including, but not limited to, a synthetic function,including, but not limited to, synthesis of proteins such as serumproteins (e.g., albumin, clotting factors, alkaline phosphatase,aminotransferases (e.g., alanine transaminase, aspartate transaminase),5′-nucleosidase, γ-glutaminyltranspeptidase, etc.), synthesis ofbilirubin, synthesis of cholesterol, and synthesis of bile acids; aliver metabolic function, including, but not limited to, carbohydratemetabolism, amino acid and ammonia metabolism, hormone metabolism, andlipid metabolism; detoxification of exogenous drugs; a hemodynamicfunction, including splanchnic and portal hemodynamics; and the like.

Drug delivery devices that are suitable for use in the subject methodsinclude, but are not limited to, injection devices; an implantabledevice, e.g., pumps, such as an osmotic pump, that may or may not beconnected to a catheter, biodegradable implants; liposomes; depots; andmicrospheres.

The term “dosing event” as used herein refers to administration of anantiviral agent to a patient in need thereof, which event may encompassone or more releases of an antiviral agent from a drug dispensingdevice. Thus, the term “dosing event,” as used herein, includes, but isnot limited to, installation of a depot comprising an antiviral agent;installation of a continuous delivery device (e.g., a pump or othercontrolled release injectible system); and a single subcutaneousinjection followed by installation of a continuous delivery system.

The term “depot” refers to any of a number of implantable, biodegradableor non-biodegradable, controlled release systems that are generallynon-containerized and that act as a reservoir for a drug, and from whichdrug is released. Depots include polymeric non-polymeric biodegradablematerials, and may be solid, semi-solid, or liquid in form.

The term “microsphere” (also referred to as “microparticles,”“nanospheres,” or “nanoparticles”) refers to small particles, generallyprepared from a polymeric material and usually having a size in therange of from about 0.01 μm to about 0.1 μm, or from about 0.1 μm toabout 10 μm in diameter.

The term “therapeutically effective amount” is meant an amount of atherapeutic agent, or a rate of delivery of a therapeutic agent,effective to facilitate a desired therapeutic effect. The precisedesired therapeutic effect will vary according to the condition to betreated, the formulation to be administered, and a variety of otherfactors that are appreciated by those of ordinary skill in the art.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “adose” includes a plurality of such doses and reference to “the method”includes reference to one or more methods and equivalents thereof knownto those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating hepatitis virusinfection, with hepatitis C virus (HCV) infection being of particularinterest. The methods generally involve administering to an individualan antiviral agent in an amount effective to reduce the viral load inthe individual, and in particular to achieve a sustained viral responsein the individual. An antiviral agent is delivered to the individual ina dosing regimen that is effective to achieve a multiphasicconcentration of the antiviral agent in the serum. The multiphasicconcentration profile of the antiviral agent is designed to take intoaccount the viral kinetics observed during treatment of hepatitis Cvirus (HCV) with IFN-α.

Currently available IFN-α therapies for treating HCV infection generallyinvolve subcutaneous injections of IFN-α daily (QD), every other day(QOD), or three times a week (TIW).

The kinetics of HCV infection among responders in response toconventional IFN-α therapies, as determined by RNA PCR, have beenanalyzed by mathematical modeling, and are shown in FIG. 1. Such studieshave clearly shown a rapid viral decline phase in 2448 hours after thebeginning of treatment, resulting in an approximately 0.5-log to anapproximately. 3-log or greater decrease in serum RNA levels. This earlyviral response (EVR) is important in reducing the production of viralparticles. An early, robust response is generally predictive of a moredurable response. This early phase is usually followed by a slower,sustained clearance of the virus over several days or weeks.

Generally, this second phase is dependent on characteristics associatedwith the patient. Without wishing to be bound by any one theory, thesecond phase reduction in viral titer may be related to removal ofvirus-infected cells, e.g., by immune system mediated mechanisms. Theslope of this second phase is determinative of the sustained viralresponse (SVR) of the patient, e.g., a steeper second phase slope isgenerally associated with a SVR and a positive treatment outcome.

Viral kinetics and serum IFN-α concentration during the course ofIFN-α-based therapy regimens are depicted in FIGS. 2-6. Viral kinetics(VK; depicted as viral RNA (“RNA”) versus time) is shown together withserum IFN-α concentration (pharmacokinetics (PK); depicted as serum IFNversus time) for both conventional (e.g., TIW) therapy and a therapeuticdosing regimen according to the invention (e.g., controlled releasetherapy, such as CRI therapy). The maximum serum concentrations of IFN-αachieved following pulsatile or repeat administrations of an activeingredient are denoted by C1max (FIGS. 2-5), C2max (FIG. 6), etc. C1max,C2max, etc., are at or near the maximum tolerated dose (MTD). The serumconcentration of IFN-α achieved over a sustained period of time isdenoted Csus (FIGS. 2-6). Csus is about 50% of the MTD. The amount ofbioavailable antiviral drug is indicated by the area under the serumconcentration versus time profile or area under the curve (AUC). Thethreshold concentration in serum when adverse effects appear is denotedthe MTD.

Current therapies to treat HCV infection suffer from certain drawbacks.Dosing regimens involving daily (QD), every other day (QOD), or thriceweekly (TIW) injections of IFN-α over extended treatment periods sufferfrom one or more of the following drawbacks: (1) the dosing regimens areuncomfortable to the patient and, in some cases, result in reducedpatient compliance; (2) the dosing regimens are often associated withadverse effects, causing additional discomfort to the patient, and, insome cases, resulting in reduced patient compliance; (3) the dosingregimens result in “peaks” (Cmax) and “troughs” (Cmin) in serum IFN-αconcentration, and, during the “trough” periods, virus can replicate,and/or infect additional cells, and/or mutate; (4) in many cases, thelog reduction in viral titer during the early viral response isinsufficient to effect a sustained viral response that ultimatelyresults in clearance of the virus (see FIG. 2; viral kinetics afterconventional IFN-α TIW therapy).

The instant invention provides dosing regimens that avoids thesedrawbacks, and provides significant advantages, including the following:(1) because the administration is less frequent than QD, QOD, or TIW,patient discomfort is reduced, which potentially increases patientcompliance; (2) because the dosing is continuous over a period of time,“peaks” (i.e., Cmax) and “toughs” (i.e., Cmin) in serum IFN-αconcentrations are avoided, e.g., the Cmax to Cmin ratio is reduced; (3)because the peak/trough cycles associated with previous dosing regimensare avoided, adverse effects are reduced; (4) because the peak/troughcycles associated with previous dosing regimens are avoided, viralreplication, infection of further cells, and mutation is reduced (i.e.,there is constant “pressure” on the virus, as there is a more constantlevel of antiviral agent in the serum); (5) one dosing event accordingto the invention addresses both the early viral response and thesustained viral responses phases of viral kinetics (see, e.g., FIG. 5,scenario III); (6) repeated dosing events according to the invention hasan effect on the sustained viral response, reducing viral titer stillfurther (see, e.g., FIG. 6: C1max, C2max, etc., exert enormous negativeselective pressure on the virus, reducing viral mutation and/orreplication and/or evasion events between dosing cycles); (7) the logreduction in viral titer during first phase of the dosing eventaccording to the invention is greater than with previously availabledosing regimens discussed above (see, e.g., FIG. 3, scenario 1; (8) theconstant high drug concentration in the sustained phase (Csus) makes thesecond phase slope steeper (see, e.g., FIG. 4, scenario H); and (9)because the log reduction in viral titer is increased, the outcomeduring the second phase is more favorable, i.e., the decrease in theviral titer during the sustained viral response phase is more rapid (theslope is steeper) than with previous dosing regimens discussed above.

The present invention provides methods of treating hepatitis viralinfection, involving a dosing regimen that provides for a multiphasicserum concentration of antiviral agent. The multiphasic serumconcentration of antiviral agent is achieved with less frequent dosingevents than with current therapies.

During a first phase, the serum concentration of IFN-α is high, toprovide optimal Cmax concentrations, and to effect as steep a slope aspossible in the viral titer, bringing the viral titer down rapidly suchthat a lower concentration of IFN-α will be effective. This initial highdose of IFN-α is referred to as the “first dose” or the “initial loadingdose”.

During a second phase, the IFN-α serum concentration is lower than inthe first phase, and is effective to reduce the viral titer stillfurther. The first phase is kept as short as possible, since the amountof IFN-α delivered during this phase is at or near the maximum dose thatis tolerated by an individual (the “MTD”). Once the viral titer isbrought down quickly during this initial, high-dosage phase, theconcentration of IFN-α can be lowered, yet achieve an AUC sufficientremain effective to reduce still further the viral titer (see, e.g.,FIG. 3). This second, lower concentration of IFN-α is tolerated by mostindividuals; thus, patient comfort and compliance is maximized.

The first phase and the second phase are achieved in a single dosingevent, e.g., where a “single dosing event” includes installation of adepot; installation of a pump; and the combination of a singlesubcutaneous injection followed by installation of a pump. A singledosing event is achieved by one or more dosage forms, e.g., one or moreof: a depot; a pump; and an injection device.

In some embodiments, antiviral agent is administered in a depot. Thisform of administration takes advantage of a property of depot deliverythat is generally considered undesirable, namely the initial “burst” ofdrug release from the depot after implantation or injection into apatient. By delivering the antiviral agent in a depot formulation thatdoes release an initial burst of antiviral agent, a multiphasic serumconcentration of antiviral agent is achieved. The initial burst ofantiviral agent release effects the first serum concentration ofantiviral agent that is effective in bringing down viral titers quickly,to a level that is treatable by a lower concentration of antiviralagent. This lower serum concentration of antiviral agent is achieved bythe sustained release of antiviral agent from the depot following theinitial burst.

In many embodiments, the dosing regimen involves a single dosing event.In other embodiments, the dosing regimen dosing event is repeated.Repeat administrations using such delivery systems provide C1max, C2max,etc., in each case followed by a steady state concentration (Csus; asshown in FIG. 6).

Methods of Treating a Hepatitis Infection

The instant invention provides methods of treating a hepatitis virusinfection. The methods generally involve administering an antiviralagent at a level and in a manner effective to achieve a multiphasicserum concentration of the antiviral agent. A first phase and a secondphase are achieved with a single dosing event (e.g., installation (e.g.,implantation or injection) of a depot; installation of a continuousinfusion device, such as a pump; a combination of a single subcutaneousinjection and installation of a continuous infusion device).

In all embodiments of the invention, the dosing regimens of the methodsof the invention achieve serum concentrations of antiviral agent inwhich the “peaks” (Cmax; the highest serum concentration of antiviralagent) and “troughs” (Cmin; the lowest serum concentration of antiviralagent) of serum antiviral agent concentration are reduced or avoided. Inall embodiments, the dosing regimens of the instant methods result-inCmax:Cmin ratio of less than about 3.0, less than about 2.5, less thanabout 2.0, or less than about 1.5 during the second phase (e.g., duringdays 2-15 of treatment, during days 2-10 of treatment, during days 3-10of treatment, or during days 3-15 of treatment, as shown in FIGS. 2-6).In some embodiments, the dosing regimens achieve a Cmax:Cmin ratio ofabout 1.0 during the second phase (e.g., during days 2-15 of treatment,during days 2-10 of treatment, during days 3-10 of treatment, or duringdays 3-15 of treatment, as shown in FIGS. 2-6).

In general, in the dosing regimens of the methods of the invention, anarea under the curve (AUC) of antiviral agent serum concentration versustime during the second phase, measured during any 24-hour period of thesecond phase, (i.e., AUC_(sus) is less than the AUC for any 24-hoursperiod of the first phase (i.e., AUC_(max)). In other words, theAUC_(sus) measured during any 24-hour period of the second phase is lessthan the AUC_(max) measured during any 24-hour period of the firstphase.

The serum concentration of antiviral agent in the first phase iseffective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a 3.5-log,a 4-log, a 4.5-log, or a 5-log reduction in viral titer in the serum ofthe individual.

The serum concentration of antiviral agent in the first phase iseffective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a 3.5-log,a 4-log, a 4.5-log, or a 5-log reduction in viral titer in the serum ofthe individual within a period of from about 12 hours to about 48 hours,or from about 16 hours to about 24 hours after the beginning of thedosing regimen.

The second concentration of antiviral agent is maintained for a periodof from about 24 hours to about 48 hours, from about 2 days to about 4days, from about 4 days to about 7 days, from about 1 week to about 2weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks toabout 12 weeks, from about 12 weeks to about 16 weeks, from about 16weeks to about 24 weeks, or from about 24 weeks to about 48 weeks.

In the second phase, the concentration of antiviral agent in the serumis effective to reduce viral titers to undetectable levels, e.g., toabout 1000 to about 5000, to about 500 to about 1000, or to about 100 toabout 500 genome copies/mL serum. In some embodiments, an effectiveamount of antiviral agent is an amount that is effective to reduce viralload to lower than 100 genome copies/mL serum.

The serum concentration of antiviral agent in the second phase iseffective to achieve a sustained viral response, e.g., no detectable HCVRNA (e.g., less than about 500, less than about 200, or less thanabout-100 genome copies per milliliter serum) is found in the patient'sserum for a period of at least about one month, at least about twomonths, at least about three months, at least about four months, atleast about five months, or at least about six months followingcessation of therapy.

In some embodiments, at least a third phase follows the first and secondphases. In some of these embodiments, third phase includes administeringantiviral agent in a dose effective to achieve a serum concentration ofantiviral agent equal or nearly equal to that of the first serumconcentration. In some of these embodiments, a fourth phase includesadministering antiviral agent in a dose effective to achieve a serumconcentration of antiviral agent equal or nearly equal to that of thesecond serum concentration.

IFN-α Treatment of HCV Infection

In certain embodiments of interest, the hepatitis virus is hepatitis Cvirus (HCV). In particular embodiments of interest, the hepatitis virusis HCV, and the antiviral agent is interferon-α (IFN-α).

In a first phase, a serum concentration of IFN-α is achieved that is ator near the maximum level that is tolerable by the patient. The serumconcentration that is achieved in the first phase (the firstconcentration) is in a range of from about 10 to about 1000, from about10 to about 500, from about 20 to about 250, from about 30 to about 100,or from about 50 to about 75 International Units (IU)/ml. The firstserum concentration is maintained for a period of from about 6 hours toabout 12 hours, from about 12 hours to about 24 hours, or from about 24hours to about 48 hours.

In the first phase, an amount of IFN-α is administered that is effectiveto achieve a serum concentration of IFN-α that is from about 65% toabout 70%, from about 70% to about 75%, from about 75% to about 80%,from about 80% to about 85%, from about 85% to about 90%, from about 90%to about 95%, or from about 95% to about 100% of the maximum tolerateddose (MTD). Thus, within a period of from about 6 hours to about 12hours, from about 12 hours to about 24 hours, or from about 24 hours toabout 48 hours from the beginning of the dosing regimen, a serumconcentration of IFN-α is achieved that is from about 65% to about 70%,from about 70% to about 75%, from about 75% to about 80%, from about 80%to about 85%, from about 85% to about 90%/, from about 90% to about 95%,or from about 95% to about 100% of the maximum tolerated dose (MTD).

The administered dose to achieve the first serum concentration of IFN-αis in a range of from about 10 μg to about 100 μg, from about 20 μg toabout 70 μg, from about 25 μg to about 60 μg, from about 30 μg to about50 μg. These various doses refer to free interferon and the amounts ofthe depots to administer to achieve this will depend on drug loadingefficiencies, as discussed below.

Effective dosages of consensus IFN-α include about 3 μg, about 9 μg,about 15 μg, about 18 μg, or about 27 μg per dose. Effective dosages ofIFN-α2a and IFN-α2b range from 3 million international units (MIU) to 10MIU per dose. Effective dosages of PEGylated IFN-α2a range from 90 to180 μg per dose. Effective dosages of PEGylated IFN-α2b range from 0.5μg/kg body weight to 1.5 μg/kg body weight per dose.

Patients with chronic hepatitis C generally have circulating virus atlevels of 10⁵-10⁷ genome copies/ml. In this first phase, the serumconcentration of IFN-α is effective to reduce HCV titer down to about5×10⁴ to about 10⁵, to about 10⁴ to about 5×10⁴, or to about 5×10³ toabout 10⁴ genome copies per milliliter serum.

In some embodiments, the serum concentration of IFN-α in the first phaseis effective to reduce HCV titer down to about 5×10⁴ to about 10⁵, toabout 10⁴ to about 5×10⁴, or to about 5×10³ to about 10⁴ genome copiesper milliliter serum within a period of from about 12 hours to about 48hours, or from about 16 hours to about 24 hours after the beginning ofthe dosing regimen.

In some embodiments, the serum concentration of IFN-α in the first phaseis effective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in viral titer in theserum of the individual.

In some embodiments, the serum concentration of IFN-α in the first phaseis effective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in viral titer in theserum of the individual within a period of from about 12 hours to about48 hours, or from about 16 hours to about 24 hours after the beginningof the dosing regimen.

In the first phase, a serum concentration of IFN-α is achieved that iseffective to reduce the viral titer to a level that is treatable with adose of interferon that can be tolerated by an infected individual.

In the second phase, IFN-α is administered at a level that is effectiveto achieve a serum concentration of IFN-α that is well below the maximumlevel that can be tolerated by the patient, and that is effective toreduce the viral titer still further. In the second phase, IFN-α isadministered at a dose that is effective to achieve a serumconcentration of IFN-α of from about 5 IU/ml to about 50 IU/ml. In someembodiments, IFN-α is administered at a dose that is effective toachieve a serum concentration of IFN-α of from about 5 IU/ml to about100 IU/ml or higher. In this second phase, the administered dose ofIFN-α is in a range of from about 0.5×10⁶ IU to about 50×10⁶ IU.

In the second phase, IFN-α is administered at a level that is effectiveto achieve and maintain a serum concentration of IFN-α that is fromabout 10% to about 15%, from about 15% to about 20%, from about 20% toabout 25%, from about 25% to about 30%, from about 30% to about 35%,from about 35% to about 40%, from about 40% to about 45%, or from about45% to about 50% of the MTD. The serum concentration of IFN-α in thesecond phase is well below the MTD, yet effective to exert and antiviraleffect. Thus, over a period of from about 48 hours to about 4 days, fromabout 48 hours to about 7 days, from about 48 hours to about 10 days, orfrom about 48 hours to about 15 days, after the beginning of the dosingregimen, a serum concentration of IFN-α is achieved (and generallymaintained) that is from about 10% to about 15%, from about 15% to about20%, from about 20% to about 25%, from about 25% to about 30%, fromabout 30% to about 35%, from about 35% to about 40%, from about 40% toabout 45%, or from about 45% to about 50% of the MTD.

The second concentration of IFN-α is maintained for a period of fromabout 24 hours to about 48 hours, from about 2 days to about 4 days,from about 4 days to about 7 days, from about 1 week to about 2 weeks,from about 2 weeks to about 4 weeks, from about 4 weeks to about 6weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks toabout 24 weeks, or from about 24 weeks to about 48 weeks.

In the second phase, the second concentration of serum IFN-α iseffective to reduce viral titers to about 1000 to about 5000, to about500 to about 1000, or to about 100 to about 500 genome copies/mL serum.In some embodiments, an effective amount of IFNα is an amount that iseffective to reduce viral load to lower than 100 genome copies/mL serum.

The second concentration of serum IFN-α is effective to achieve asustained viral response, e.g., no detectable HCV RNA (e.g., less thanabout 500, less than about 200, or less than about 100 genome copies permilliliter serum) is found in the patient's serum for a period of atleast about one month, at least about two months, at least about threemonths, at least about four months, at least about five months, or atleast about six months following cessation of therapy.

In some embodiments, at least a third phase follows the first and secondphases. In some of these embodiments, third phase includes administeringIFN-α in a dose effective to achieve a serum concentration of IFN-αequal or nearly equal to that of the first serum concentration. In someof these embodiments, a fourth phase includes administering IFN-α in adose effective to achieve a serum concentration of IFN-α equal or nearlyequal to that of the second serum concentration.

Combination Therapies

In some embodiments, the methods provide for combination therapycomprising administering IFN-α and an additional therapeutic agent suchas IFN-γ and/or ribavirin. In all embodiments in which the dosingregimen comprises administration of IFN-α and an additional agent suchas IFN-γ and/or ribavirin, IFN-α is administered such that a multiphasicserum concentration of IFN-α is achieved, as described above.

In some embodiments, the additional therapeutic agent(s) is administeredduring the entire course of IFN-α treatment, and the beginning and endof the treatment periods coincide. In other embodiments, the additionaltherapeutic agent(s) is administered for a period of time that isoverlapping with that of the IFN-α treatment, e.g., treatment with theadditional therapeutic agent(s) begins before the IFN-α treatment beginsand ends before the IFN-α treatment ends; treatment with the additionaltherapeutic agent(s) begins after the IFN-α treatment begins and endsafter the IFN-γ treatment ends; treatment with the additionaltherapeutic agent(s) begins after the IFN-α treatment begins and endsbefore the IFN-α treatment ends; or treatment with the additionaltherapeutic agent(s) begins before the IFN-α treatment begins and endsafter the IFN-α treatment ends.

In still other embodiments, the additional therapeutic agent(s) isadministered before the IFN-α treatment begins, and ends once IFN-αtreatment begins, e.g., the additional therapeutic agent is used in a“priming” dosing regimen.

IFN-α and IFN-γ

In some embodiments, interferon gamma (IFN-γ) is administered separatelyfrom IFN-α, e.g., the IFN-γ is administered in a separate formulationand in a separate dosing event from IFN-α. In other embodiments, IFN-γis administered in the same formulation with IFN-α (and therefore in thesame dosing event). In still other embodiments, IFN-γ is administered ina separate formulation from IFN-α, and is administered in a dosingregimen that provides for a multiphasic serum concentration as describedabove.

Effective dosages of IFN-γ range from about 0.5 μg/m² to about 500μg/m², usually from about 1.5 μg/m² to 200 μg/m², depending on the sizeof the patient. This activity is based on 10⁶ international units (IU)per 50 μg of protein.

As noted above, in some embodiments, IFN-γ is administered in a separatedosing event, as IFN-α. In one non-limiting example, IFN-γ isadministered in a dose of about 1 MIU/day for 14 days; followed by 5MIU/day for 14 days; followed by 5MIU three times per week for 22 weeks.

In some embodiments, IFN-γ is administered during the entire course ofIFN-α treatment. In other embodiments, IFN-γ is administered for aperiod of time that is overlapping with that of the IFN-α treatment,e.g., the IFN-γ treatment can begin before the IFN-α treatment beginsand end before the IFN-α treatment ends; the IFN-γ treatment can beginafter the IFN-α treatment begins and end after the IFN-γ treatment ends;the IFN-γ treatment can begin after the IFN-α treatment begins and endbefore the IFN-α treatment ends; or the IFN-γ treatment can begin beforethe IFN-α treatment begins and end after the IFN-α treatment ends.

In some embodiments, IFN-γ is administered for a period of time beforeIFN-α is administered. Without wishing to be bound by any one theory,IFN-γ may effect a Th2 to Th1 shift. This increase in a Th1 immuneresponse may result in an increase in the rate of reduction of viraltiter once IFN-α administration is initiated. In these embodiments,IFN-γ is administered for a period of time from about 1 day to about 14days, from about 2 days to about 10 days, or from about 3 days to about7 days, before the beginning of treatment with IFN-α. This period oftime is referred to as the “priming” phase. In some of theseembodiments, IFN-γ treatment is continued throughout the entire periodof treatment with IFN-α. In other embodiments, IFN-γ treatment isdiscontinued before the end of treatment with IFN-α. In theseembodiments, the total time of treatment with IFN-γ (including the“priming” phase) is from about 2 days to about 30 days, from about 4days to about 25 days, from about 8 days to about 20 days, from about 10days to about 18 days, or from about 12 days to about 16 days.

IFN-γ can be administered by any conventional route and means,including, but not limited to, subcutaneously, intradermally, orallyletc. IFN-γ can also be administered by the methods of the invention,providing for multiphasic serum concentration of IFN-γ. Administrationcan be by injection, by a continuous infusion device (e.g., a pump), andthe like. In many embodiments, IFN-γ is administered subcutaneously byinjection.

IFN-α and Ribavirin

Ribavirin, 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide,available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif., isdescribed in the Merck Index, compound No. 8199, Eleventh Edition. Itsmanufacture and formulation is described in U.S. Pat. No. 4,211,771. Theinvention also contemplates use of derivatives of ribavirin (see, e.g.,U.S. Pat. No. 6,277,830). The ribavirin may be administered orally incapsule or tablet form, or in the same or different administration formand in the same or different route as the IFN-α. Of course, other typesof administration of both medicaments, as they become available arecontemplated, such as by nasal spray, transdermally, intravenously, bysuppository, by sustained release dosage form, etc. Any form ofadministration will work so long as the proper dosages are deliveredwithout destroying the active ingredient.

Ribavirin is generally administered in an amount ranging from about 30mg to about 60 mg, from about 60 mg to about 125 mg, from about 125 mgto about 200 mg, from about 200 mg to about 300 gm, from about 300 mg toabout 400 mg, from about 400 mg to about 1200 mg, from about 600 mg toabout 1000 mg, or from about 700 to about 900 mg per day.

In some embodiments, ribavirin is administered throughout the entirecourse of IFN-α treatment. In other embodiments, ribavirin isadministered less than the entire course of IFN-α treatment, e.g., onlyduring the first phase of IFN-α treatment, only during the second phaseof IFN-α treatment, or some other portion of the IFN-α treatmentregimen.

Antiviral Agents

Any of a variety of antiviral agents can be delivered using the methodsof the invention. Antiviral agents suitable for use in the instantmethods include, but are not limited to, IFN-α, IFN-γ, and ribavirin.

IFN-Alpha

Any known IFN-α can be used in the instant invention. The term“interferon-alpha” as used herein refers to a family of relatedpolypeptides that inhibit viral replication and cellular proliferationand modulate immune response. The term “IFN-α” includes naturallyoccurring IFN-α; synthetic IFN-α; derivatized IFN-α (e.g., PEGylatedIFN-α, glycosylated IFN-α, and the like); and analogs of naturallyoccurring or synthetic IFN-α; essentially any IFN-α that has antiviralproperties, as described for naturally occurring IFN-α.

Suitable alpha interferons include, but are not limited to,naturally-occurring IFN-α (including, but not limited to, naturallyoccurring IFN-α2a, IFN-α2b); recombinant interferon alpha-2b such asIntron®A interferon available from Schering Corporation, Kenilworth,N.J.; recombinant interferon alpha-2a such as Roferon® interferonavailable from Hoffmann-La Roche, Nutley, N.J.; recombinant interferonalpha-2C such as Berofor® alpha 2 interferon available from BoehringerIngelheim Pharmaceutical, Inc., Ridgefield, Conn.; interferon alpha-n1,a purified blend of natural alpha interferons such as Sumiferonavailable from Sumitomo, Japan or as Wellferon® interferon alpha-n1(INS) available from the Glaxo-Wellcome Ltd., London, Great Britain; andinterferon alpha-n3 a mixture of natural alpha interferons made byInterferon Sciences and available from the Purdue Frederick Co.,Norwalk, Conn., under the Alferon® Tradename.

The term “IFN-α” also encompasses consensus IFN-α. Consensus IFN-α (alsoreferred to as “CIFN” and “IFN-con”) encompasses but is not limited tothe amino acid sequences designated IFN-con₁, IFN-con₂ and IFN-con₃which are disclosed in U.S. Pat. Nos. 4,695,623 and 4,897,471; andconsensus interferon as defined by determination of a consensus sequenceof naturally occurring interferon alphas (e.g., Infergen®, Amgen,Thousand Oaks, Calif.). DNA sequences encoding IFN-con may besynthesized as described in the aforementioned patents or other standardmethods. Use of CIFN is of particular interest.

The term “IFN-α” also encompasses derivatives of IFN-α that arederivatized (e.g., are chemically modified) to alter certain propertiessuch as serum half-life. As such, the term “IFN-α” includes glycosylatedIFN-α; IFN-α derivatized with polyethylene glycol (“PEGylated IFN-α”);and the like. PEGylated IFN-α, and methods for making same, is discussedin, e.g., U.S. Pat. Nos. 5,382,657; 5,981,709; and 5,951,974. PEGylatedIFN-α encompasses conjugates of PEG and any of the above-described IFN-αmolecules, including, but not limited to, PEG conjugated to interferonalpha-2a (Roferon, Hoffman La-Roche, Nutley, N.J.), interferon alpha 2b(Intron, Schering-Plough, Madison, N.J.), interferon alpha-2c (BeroforAlpha, Boehringer Ingelheim, Ingelheim, Germany); and consensusinterferon as defined by determination of a consensus sequence ofnaturally occurring interferon alphas (infergen, Amgen, Thousand Oaks,Calif.).

Interferon-Gamma

The nucleic acid sequences encoding IFN-γ polypeptides may be accessedfrom public databases, e.g., Genbank, journal publications, etc. Whilevarious mammalian IFN-γ polypeptides are of interest, for the treatmentof human disease, generally the human protein will be used. Human IFN-γcoding sequence may be found in Genbank, accession numbers X13274;V00543; and NM_(—)000619. The corresponding genomic sequence may befound in Genbank, accession numbers J00219; M37265; and V00536. See, forexample. Gray et al. (1982) Nature 295:501 (Genbank X13274); andRinderknecht et al. (1984) J.B.C. 259:6790.

IFN-γ1b (Actimmune®; human interferon) is a single-chain polypeptide of140 amino acids. It is made recombinantly in E.coli and isunglycosylated. Rinderknecht et al. (1984) J. Biol. Chem. 259:6790-6797.

The IFN-γ to be used in the methods of the present invention may be anyof natural IFN-γs, recombinant IFN-γs and the derivatives thereof so faras they have an IFN-γ activity, particularly human IFN-γ activity. HumanIFN-γ exhibits the antiviral and anti-proliferative propertiescharacteristic of the interferons, as well as a number of otherimmunomodulatory activities, as is known in the art. Although IFN-γ isbased on the sequences as provided above, the production of the proteinand proteolytic processing can result in processing variants thereof.The unprocessed sequence provided by Gray et al., supra, consists of 166amino acids (aa). Although the recombinant IFN-γ produced in E. coli wasoriginally believed to be 146 amino acids, (commencing at amino acid 20)it was subsequently found that native human IFN-1 is cleaved afterresidue 23, to produce a 143 aa protein, or 144 aa if the terminalmethionine is present, as required for expression in bacteria. Duringpurification, the mature protein can additionally be cleaved at the Cterminus after reside 162 (referring to the Gray et al. sequence),resulting in a protein of 139 amino acids, or 140 amino acids if theinitial methionine is present, e.g. if required for bacterialexpression. The N-terminal methionine is an artifact encoded by the mRNAtranslational “start” signal AUG that, in the particular case of E. coliexpression is not processed away. In other microbial systems oreukaryotic expression systems, methionine may be removed.

For use in the subject methods, any of the native IFN-γ peptides,modifications and variants thereof, or a combination of one or morepeptides may be used. IFN-γ peptides of interest include fragments, andcan be variously truncated at the carboxy terminal end relative to thefull sequence. Such fragments continue to exhibit the characteristicproperties of human gamma interferon, so long as amino acids 24 to about149 (numbering from the residues of the unprocessed polypeptide) arepresent. Extraneous sequences can be substituted for the amino acidsequence following amino acid 155 without loss of activity. See, forexample, U.S. Pat. No. 5,690,925. Native IFN-γ moieties includemolecules variously extending from amino acid residues 24-150; 24-151,24-152; 24-153, 24-155; and 24-157. Any of these variants, and othervariants known in the art and having IFN-γ activity, may be used in thepresent methods.

The sequence of the IFN-γ polypeptide may be altered in various waysknown in the art to generate targeted changes in sequence. A variantpolypeptide will usually be substantially similar to the sequencesprovided herein, i.e., will differ by at least one amino acid, and maydiffer by at least two but not more than about ten amino acids. Thesequence changes may be substitutions, insertions or deletions. Scanningmutations that systematically introduce alanine, or other residues, maybe used to determine key amino acids. Specific amino acid substitutionsof interest include conservative and non-conservative changes.Conservative amino acid substitutions typically include substitutionswithin the following groups: (glycine, alanine); (valine, isoleucine,leucine); (aspartic acid, glutamic acid); (asparagine, glutamine);(serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that may or may not alter the primary aminoacid sequence include chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation; changes in amino acid sequence thatintroduce or remove a glycosylation site; changes in amino acid sequencethat make the protein susceptible to PEGylation; and the like. Alsoincluded are modifications of glycosylation, e.g., those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g., byexposing the polypeptide to enzymes that affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.,phosphotyrosine, phosphoserine, or phosphothreonine.

Included in the subject invention are polypeptides that have beenmodified using ordinary chemical techniques so as to improve theirresistance to proteolytic degradation, to optimize solubilityproperties, or to render them more suitable as a therapeutic agent. Forexamples, the backbone of the peptide may be cyclized to enhancestability (see Friedler et al. (2000) J. Biol. Chem. 275:23783-23789).Analogs may be used that include residues other than naturally occurringL-amino acids, e.g., D-amino acids or non-naturally occurring syntheticamino acids. The protein may be pegylated to enhance stability.

The polypeptides may be prepared by in vitro synthesis, usingconventional methods as known in the art, by recombinant methods, or maybe isolated from cells induced or naturally producing the protein. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like. If desired,various groups may be introduced into the polypeptide during synthesisor during expression, which allow for linking to other molecules or to asurface. Thus cysteines can be used to make thioethers, histidines forlinking to a metal ion complex, carboxyl groups for forming amides oresters, amino groups for forming amides, and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

Ribavirin

Ribavirin, 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide,available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif., isdescribed in the Merck Index, compound No. 8199, Eleventh Edition. Itsmanufacture and formulation is described in U.S. Pat. No. 4,211,771. Theinvention also contemplates use of derivatives of ribavirin (see, e.g.,U.S. Pat. No. 6,277,830).

Liver Targeting Systems

Antiviral agents described herein can be targeted to the liver, usingany known targeting means. Those skilled in the art are aware of a widevariety of compounds that have been demonstrated to target compounds tohepatocytes. Such liver targeting compounds include, but are not limitedto, asialoglycopeptides; basic polyamino acids conjugated with galactoseor lactose residues; galactosylated albumin;asialoglycoprotein-poly-L-lysine) conjugates; lactosaminated albumin;lactosylated albumin-poly-L-lysine conjugates; galactosylatedpoly-L-lysine; galactose-PEG-poly-L-lysine conjugates;lactose-PEG-poly-L-lysine conjugates; asialofetuin; and lactosylatedalbumin.

In some embodiments, a liver targeting compound is conjugated directlyto the antiviral agent. In other embodiments, a liver targeting compoundis conjugated indirectly to the antiviral agent, e.g., via a linker. Instill other embodiments, a liver targeting compound is associated with adelivery vehicle, e.g., a liposome or a microsphere, forming ahepatocyte targeted delivery vehicle, and the antiviral agent isdelivered using the hepatocyte targeted delivery vehicle.

The terms “targeting to the liver” and “hepatocyte targeted” refer totargeting of an antiviral agent to a hepatocyte, such that at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, or at least about 90%, or more,of the antiviral agent administered to the subject enters the liver viathe hepatic portal and becomes associated with (e.g., is taken up by) ahepatocyte.

Drug Delivery Systems

Any known delivery system that is capable of providing a multiphasicserum concentration profile of antiviral agent can be used in thepresent invention. In addition, a combination of any known deliverysystem can be used.

The drug delivery system can be any device, including an implantabledevice, which device can be based on, for example, mechanical infusionpumps, electromechanical infusion pumps, depots, microspheres.Essentially, and drug delivery system that provides for controlledrelease as described above (at least biphasic release) is suitable foruse in the instant invention. In some embodiments, the drug deliverysystem is a depot. In other embodiments, the drug delivery system is acontinuous delivery device (e.g., an injectable system, a pump, etc.).In still other embodiments, the drug delivery system is a combination ofa injection device (e.g., a syringe and needle) and a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

In some embodiments, the delivery system is a depot system. Depotsystems comprise a matrix in which the IFN-α or other antiviral agent isembedded. The matrix is a polymeric or non-polymeric substance.

In certain embodiments, drug delivery system comprises a depot.

In some embodiments, the depot comprises a polymeric matrix. Forexample, a polymeric matrix derived from copolymeric and homopolymericpolyesters having hydrolysable ester linkages may be used. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Non-limiting examples ofsuch polymers are polyglycolic adds (PGA) and polylactic acids (PLA),poly(DL-lactic acid-co-glycolic acid)(DL PLGA), poly(D-lacticacid-coglycolic acid)(D PLGA) and poly(L-lactic acid-co-glycolic acid)(LPLGA). Exemplary ratios for lactic acid and glycolic acid polymers inpoly(lactic acid-co-glycolic acid) is in the range of 100:0 (i.e. purepolylactide) to 50:50. Other useful biodegradable or bioerodablepolymers include but are not limited to such polymers aspoly(ε-caprolactone), poly(ε-caprolactone-CO-lactic add), poly(ε-caprolactone-CO-glycolic acid), poly(β-hydroxy butyric acid),poly(alkyl-2-cyanoacrilate), hydrogels such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (i.e. L-leucine, glutamicacid, L-aspartic acid and the like), poly (ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters,polycarbonate, polymaleamides, polysaccharides and copolymers thereof.

In some embodiments, the drug delivery system is a poly (lacticacid-co-glycolic acid) system. Such systems are described in theliterature, e.g., in U.S. Pat. Nos. 6,183,781; and 5,654,008.

In some of these embodiments, the depot is a high viscosity liquid suchas a non-polymeric non-water-soluble liquid carrier material, e.g.,Sucrose Acetate Isobutyrate (SAIB) or another compound such as acompound described in U.S. Pat. Nos. 5,968,542; and 5,747,058. Forexample, the SABER™ system (Southern Biosystems, Inc.) is used.

Release modifying agents and/or additives can be included in the depotmatrix.

The term “release modifying agent”, as used herein, refers to a materialwhich, when incorporated into a polymer/drug matrix, modifies thedrug-release characteristics of the matrix. A release modifying agentcan, for example, either decrease or increase the rate of drug releasefrom the matrix. One group of release modifying agents includesmetal-containing salts.

One category of additives includes biodegradable polymers and oligomers.The polymers can be used to alter the release profile of the substanceto be delivered, to add integrity to the composition, or to otherwisemodify the properties of the composition. Non-limiting examples ofsuitable biodegradable polymers and oligomers include: poly(lactide),poly(lactide-co-glycolide), poly(glycolide), poly(caprolactone),polyamides, polyanhydrides, polyamino acids, polyorthoesters,polycyanoacrylates, poly(phosphazines), poly(phosphoesters),polyesteramides, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, degradable polyurethanes,polyhydroxybuty-ates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), chitin, chitosan, andcopolymers, terpolymers, oxidized cellulose, or combinations or mixturesof the above materials.

Examples of poly(α-hydroxy acid)s include poly(glycolic acid),poly(DL-lactic acid) and poly(L-lactic acid), and their copolymers.Examples of polylactones include poly(ε-caprolactone),poly(δ-valerolactone) and poly(γ-butyrolactone).

Other additives include non-biodegradable polymers. Non-limitingexamples of non-erodible polymers which can be used as additivesinclude: polyacrylates, ethylene-vinyl acetate polymers, cellulose andcellulose derivatives, acyl substituted cellulose acetates andderivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinylchloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonatedpolyolefins, and polyethylene oxide.

A further class of additives which can be used in the presentcompositions are natural and synthetic oils and fats. Oils derived fromanimals or from plant seeds of nuts typically include glycerides of thefatty acids, chiefly oleic, palmitic, stearic, and linolenic.

Other additives include film property modifying agents and releasecontrolling agents. Examples of film property modifying agents includeplasticizers, e.g. triethyl-citrate, triacetin, polyethyleneglycol,polyethyleneoxide etc. Examples of release-controlling agents includeinorganic bases (e.g. sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium carbonate, etc), organic bases (e.g. ethanol amine,diethanole amine, triethanole amine, lidocaine, tetracaine, etc,),inorganic acids (e.g. ammonium sulfate, ammonium chloride, etc), organicacids (e.g. citric acid, lactic acid, glycolic acid, ascorbic acid,etc), and solid soluble substances which upon release create pores inthe coating (e.g. crystals of sodium chloride, glucose, mannitol,sucrose, etc).

In some embodiments, the drug delivery system is a polyethyleneglycol-poly(lactic co-glycolic) acid (PEG-PLGA) based aqueous injectiblethermosensitive gel, as described in, e.g., U.S. Pat. Nos. 6,201,071;6,117,949; and 6,004,573. For example, the depot can comprise a watersoluble, biodegradable ABA- or BAB-type tri-block polymer is disclosedthat is made up of a major amount of a hydrophobic A polymer block madeof a biodegradable polyester and aminor amount of a hydrophilic PEG Bpolymer block, having an overall average molecular weight of betweenabout 2000 and 4990, and that possesses reverse thermal gelationproperties. Such materials form a gel depot within the body, from whichthe drugs are released at a controlled rate.

In some embodiments, the drug delivery system is a polyamino acid-basedsystem, e.g., as described in U.S. Pat. Nos. 6,071,538; 6,245,359;6,221,367; and 6,099,856.

In other embodiments, the drug delivery system is a microsphere.Microspheres are amply described in the literature.

In another embodiments, the drug delivery system is a pump, e.g., animplantable pump, particularly an adjustable implantable pump. Ofparticular interest is the use of an adjustable pump, particularly apump that is adjustable while in position for delivery (e.g., externallyadjustable from outside the patient's body. Such pumps includeprogrammable pumps that are capable of providing high concentrations ofIFN-α or other antiviral agent over extended periods of time, e.g.,24-72 hours, and to achieve AUC serum IFN-α concentrations to betherapeutically effective.

In some embodiments, the delivery device is a Medipad® device (ElanPharm Int'l. Ltd.).

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present invention. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852, and the like. In general, the presentmethods of drug delivery can be accomplished using any of a variety ofrefillable, pump systems. Pumps provide consistent, controlled releaseover time.

In a preferred embodiment, the drug delivery system is an at leastpartially implantable device. The implantable device can be implanted atany suitable implantation site using methods and devices well known inthe art. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are generally preferred because ofconvenience in implantation and removal of the drug delivery device.

As noted above, a combination of delivery systems can be used. As onenon-limiting example, a PLGA based system which has an initial drugrelease or burst characteristic is combined with a sucrose acetateisobutyrate based system with no drug release as a burst may be combinedtogether to achieve the desired profiles taught by this invention. Asanother non-limiting example, a loading dose such as a bolus followed bya zero-order throughput as realized or achieved with a device system.The delivery molecule may be an alpha interferon or a PEG derivatizedalpha interferon with all these delivery systems.

Depending on the drug delivery system, IFN-α can be administered orally,subcutaneously, intramuscularly, parenterally, or by other routes suchas transdermally, cutaneously, etc. There could be a burst of the drugwhen administered by such routes e.g., orally except that the drugenters portal circulation as in oral delivery and therefore of utilityin targeting the drug to the desired organ, namely liver.

In many embodiments, the IFN-α is delivered subcutaneously.

IFN-α is administered to individuals in a formulation with apharmaceutically acceptable excipient(s). A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy”, 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds7^(th) ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

IFN-α can be administered together with (i.e., simultaneously inseparate formulations; simultaneously in the same formulation;administered in separate formulations and within about 48 hours, withinabout 36 hours, within about 24 hours, within about 16 hours, withinabout 12 hours, within about 8 hours, within about 4 hours, within about2 hours, within about 1 hour, within about 30 minutes, or within about15 minutes or less) one or more additional therapeutic agents.

In other embodiments, patients are treated with a combination of IFN-αand ribavirin. Ribavirin,1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICNPharmaceuticals, Inc., Costa Mesa, Calif., is described in the MerckIndex, compound No. 8199, Eleventh Edition. Its manufacture andformulation is described in U.S. Pat. No. 4,211,771. The ribavirin maybe administered orally in capsule or tablet form in association with theadministration of IFN-α. Of course, other types of administration ofboth medicaments, as they become available are contemplated, such as bynasal spray, transdermally, intravenous, by suppository, by sustainedrelease dosage form, etc. Any form of administration will work so longas the proper dosages are delivered without destroying the activeingredient. If administered, ribavirin is administered in an amountranging from about 400 mg to about 1200 mg, from about 600 mg to about1000 mg, or from about 700 to about 900 mg per day.

In some embodiments, the combination therapy comprises IFN-α and IFN-γ.In some of these embodiments, IFN-α and IFN-γ are administered in thesame formulation, and are administered simultaneously. In otherembodiments, IFN-α and IFN-γ are administered separately, e.g., inseparate formulations. In some of these embodiments, IFN-α and IFN-γ areadministered separately, and are administered simultaneously. In otherembodiments, IFN-α and IFN-γ are administered separately and areadministered within about 5 seconds to about 15 seconds, within about 15seconds to about 30 seconds, within about 30 seconds to about 60seconds, within about 1 minute to about 5 minutes, within about 5minutes to about 15 minutes, within about 15 minutes to about 30minutes, within about 30 minutes to about 60 minutes, within about 1hour to about 2 hours, within about 2 hours to about 6 hours, withinabout 6 hours to about 12 hours, within about 12 hours to about 24hours, or within about 24 hours to about 48 hours of one another.

Determining Effectiveness of Treatment

Whether a subject method is effective in treating a hepatitis virusinfection, particularly an HCV infection, can be determined by measuringviral load, or by measuring a parameter associated with HCV infection,including, but not limited to, liver fibrosis.

Viral load can be measured by measuring the titer or level of virus inserum. These methods include, but are not limited to, a quantitativepolymerase chain reaction (PCR) and a branched DNA (bDNA) test. Forexample, quantitative assays for measuring the viral load (titer) of HCVRNA have been developed. Many such assays are available commercially,including a quantitative reverse transcription PCR (RT-PCR) (AmplicorHCV Monitor™, Roche Molecular Systems, New Jersey); and a branched DNA(deoxyribonucleic acid) signal amplification assay (Quantiplex™ HCV RNAAssay (bDNA), Chiron Corp., Emeryville, Calif.). See, e.g., Gretch etal. (1995) Ann. Intern. Med. 123:321-329.

As noted above, whether a subject method is effective in treating ahepatitis virus infection, e.g., an HCV infection, can be determined bymeasuring a parameter associated with hepatitis virus infection, such asliver fibrosis. Liver fibrosis reduction is determined by analyzing aliver biopsy sample. An analysis of a liver biopsy comprises assessmentsof two major components: necroinflammation assessed by “grade” as ameasure of the severity and ongoing disease activity, and the lesions offibrosis and parenchymal or vascular remodeling as assessed by “stage”as being reflective of long-term disease progression. See, e.g., Brunt(2000) Hepatol. 31:241-246; and METAVIR (1994) Hepatology 20:15-20.Based on analysis of the liver biopsy, a score is assigned. A number ofstandardized scoring systems exist which provide a quantitativeassessment of the degree and severity of fibrosis. These include theMETAVIR, Knodell, Scheuer, Ludwig, and Ishak scoring systems.

Serum markers of liver fibrosis can also be measured as an indication ofthe efficacy of a subject treatment method. Serum markers of liverfibrosis include, but are not limited to, hyaluronate, N-terminalprocollagen III peptide, 7S domain of type IV collagen, C-terminalprocollagen I peptide, and laminin. Additional biochemical markers ofliver fibrosis include α-2-macroglobulin, haptoglobin, gamma globulin,apolipoprotein A, and gamma glutamyl transpeptidase.

As one non-limiting example, levels of serum alanine aminotransferase(ALT) are measured, using standard assays. In general, an ALT level ofless than about 45 international units per milliliter serum isconsidered normal. In some embodiments, an effective amount of IFNα isan amount effective to reduce ALT levels to less than about 45 IU/mlserum.

Methods of Treating Liver Fibrosis

The present invention provides methods of treating liver fibrosis. Themethods involve administering an antiviral agent, as describe above,wherein viral load is reduced in the individual, and wherein liverfibrosis is treated. Treating liver fibrosis includes reducing the riskthat liver fibrosis will occur; reducing a symptom associated with liverfibrosis; and increasing liver function.

Whether treatment with antiviral agent as described herein is effectivein reducing liver fibrosis is determined by any of a number ofwell-established techniques for measuring liver fibrosis and liverfunction. Liver fibrosis reduction is determined by analyzing a liverbiopsy sample. An analysis of a liver biopsy comprises assessments oftwo major components: necroinflammation assessed by “grade” as a measureof the severity and ongoing disease activity, and the lesions offibrosis and parenchymal or vascular remodeling as assessed by “stage”as being reflective of long-term disease progression. See, e.g., Brunt(2000) Hepatol. 31:241-246; and METAVIR (1994) Hepatology 20:15-20.Based on analysis of the liver biopsy, a score is assigned. A number ofstandardized scoring systems exist which provide a quantitativeassessment of the degree and severity of fibrosis. These include theMETAVIR, Knodell, Scheuer, Ludwig, and Ishak scoring systems.

The METAVIR scoring system is based on an analysis of various featuresof a liver biopsy, including fibrosis (portal fibrosis, centrilobularfibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis,acidophilic retraction, and ballooning degeneration); inflammation(portal tract inflammation, portal lymphoid aggregates, and distributionof portal inflammation); bile duct changes; and the Knodell index(scores of periportal necrosis, lobular necrosis, portal inflammation,fibrosis, and overall disease activity). The definitions of each stagein the METAVIR system are as follows: score: 0, no fibrosis; score: 1,stellate enlargement of portal tract but without septa formation; score:2, enlargement of portal tract with rare septa formation; score: 3,numerous septa without cirrhosis; and score: 4, cirrhosis.

Knodell's scoring system, also called the Hepatitis Activity Index,classifies specimens based on scores in four categories of histologicfeatures: I. Periportal and/or bridging necrosis; 11. Intralobulardegeneration and focal necrosis; 111. Portal inflammation; and IV.Fibrosis. In the Knodell staging system, scores are as follows: score:0, no fibrosis; score: I, mild fibrosis (fibrous portal expansion);score: 2, moderate fibrosis; score: 3, severe fibrosis (bridgingfibrosis); and score: 4, cirrhosis. The higher the score, the moresevere the liver tissue damage. Knodell (1981) Hepatol. 1:431.

In the Scheuer scoring system scores are as follows: score: 0, nofibrosis; score: 1, enlarged, fibrotic portal tracts; score: 2,periportal or portal-portal septa, but intact architecture; score: 3,fibrosis with architectural distortion, but no obvious cirrhosis; score:4, probable or definite cirrhosis. Scheuer (1991) J. Hepatol. 13:372.

The Ishak scoring system is described in Ishak (1995) J. Hepatol.22:696-699. Stage O, No fibrosis; Stage 1, Fibrous expansion of someportal areas, with or without short fibrous septa; stage 2, Fibrousexpansion of most portal areas, with or without short fibrous septa;stage 3, Fibrous expansion of most portal areas with occasional portalto portal (P-P) bridging; stage 4, Fibrous expansion of portal areaswith marked bridging (P-P) as well as portal-central (P-C); stage 5,Marked bridging (P-P and/or P-C) with occasional nodules (incompletecirrhosis); stage 6, Cirrhosis, probable or definite. The benefit ofanti-fibrotic therapy can also be measured and assessed by using theChild-Pugh scoring system which comprises a multicomponent point systembased upon abnormalities in serum bilirubin level, serum albumin level,prothrombin time, the presence and severity of ascites, and the presenceand severity of encephalopathy. Based upon the presence and severity ofabnormality of these parameters, patients may be placed in one of threecategories of increasing severity of clinical disease: A, B, or C.

In some embodiments, a therapeutically effective amount of antiviralagent is an amount of antiviral agent that effects a change of one unitor more in the fibrosis stage based on pre-and post-therapy liverbiopsies. In particular embodiments, a therapeutically effective amountof IFN-α and IFN-γ reduces liver fibrosis by at least one unit in theMETAVIR, the Knodell, the Scheuer, the Ludwig, or the Ishak scoringsystem.

Secondary, or indirect, indices of liver function can also be used toevaluate the efficacy of treatment. Morphometric computerizedsemi-automated assessment of the quantitative degree of liver fibrosisbased upon specific staining of collagen and/or serum markers of liverfibrosis can also be measured as an indication of the efficacy of asubject treatment method. Secondary indices of liver function include,but are not limited to, serum transaminase levels, prothrombin time,bilirubin, platelet count, portal pressure, albumin level, andassessment of the Child-Pugh score. An effective amount of antiviralagent is an amount that is effective to increase an index of liverfunction by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, or at leastabout 80%, or more, compared to the index of liver function in anuntreated individual, or to a placebo-treated individual. Those skilledin the art can readily measure such indices of liver function, usingstandard assay methods, many of which are commercially available, andare used routinely in clinical settings.

Serum markers of liver fibrosis can also be measured as an indication ofthe efficacy of a subject treatment method. Serum markers of liverfibrosis include, but are not limited to, hyaluronate, N-terminalprocollagen III peptide, 7S domain of type IV collagen, C-terminalprocollagen I peptide, and laminin. Additional biochemical markers ofliver fibrosis include α-2-macroglobulin, haptoglobin, gamma globulin,apolipoprotein A, and gamma glutamyl transpeptidase.

A therapeutically effective amount of antiviral agent is an amount thatis effective to reduce a serum level of a marker of liver fibrosis by atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, or at least about 80%, ormore, compared to the level of the marker in an untreated individual, orto a placebo-treated individual. Those skilled in the art can readilymeasure such serum markers of liver fibrosis, using standard assaymethods, many of which are commercially available, and are usedroutinely in clinical settings. Methods of measuring serum markersinclude immunological-based methods, e.g., enzyme-linked immunosorbentassays (ELISA), radioimmunoassays, and the like, using antibody specificfor a given serum marker.

Quantitative tests of functional liver reserve can also be used toassess the efficacy of treatment with antiviral agent. These include:indocyanine green clearance (ICG), galactose elimination capacity (GEC),aminopyrine breath test (ABT), antipyrine clearance,monoethylglycine-xylidide (MEG-X) clearance, and caffeine clearance.

As used herein, a “complication associated with cirrhosis of the liver”refers to a disorder that is a sequellae of decompensated liver disease,i.e., or occurs subsequently to and as a result of development of liverfibrosis, and includes, but it not limited to, development of ascites,variceal bleeding, portal hypertension, jaundice, progressive liverinsufficiency, encephalopathy, hepatocellular carcinoma, liver failurerequiring liver transplantation, and liver-related mortality.

A therapeutically effective amount of antiviral agent is an amount thatis effective in reducing the incidence (e.g., the likelihood that anindividual will develop) of a disorder associated with cirrhosis of theliver by at least about 10%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, or at leastabout 80%, or more, compared to an untreated individual, or to aplacebo-treated individual.

Whether treatment with antiviral agent is effective in reducing theincidence of a disorder associated with cirrhosis of the liver canreadily be determined by those skilled in the art.

Reduction in liver fibrosis increases liver function. Thus, theinvention provides methods for increasing liver function, generallyinvolving administering a therapeutically effective amount of antiviralagent. Liver functions include, but are not limited to, synthesis ofproteins such as serum proteins (e.g., albumin, clotting factors,alkaline phosphatase, aminotransferases (e.g., alanine transaminase,aspartate transaminase), 5′-nucleosidase, γ-glutaminyltranspeptidase,etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesisof bile acids; a liver metabolic function, including, but not limitedto, carbohydrate metabolism, amino acid and ammonia metabolism, hormonemetabolism, and lipid metabolism; detoxification of exogenous drugs; ahemodynamic function, including splanchnic and portal hemodynamics; andthe like.

Whether a liver function is increased is readily ascertainable by thoseskilled in the art, using well-established tests of liver function.Thus, synthesis of markers of liver function such as albumin, alkalinephosphatase, alanine transaminase, aspartate transaminase, bilirubin,and the like, can be assessed by measuring the level of these markers inthe serum, using standard immunological and enzymatic assays. Splanchniccirculation and portal hemodynamics can be measured by portal wedgepressure and/or resistance using standard methods. Metabolic functionscan be measured by measuring the level of ammonia in the serum.

Whether serum proteins normally secreted by the liver are in the normalrange can be determined by measuring the levels of such proteins, usingstandard immunological and enzymatic assays. Those skilled in the artknow the normal ranges for such serum proteins. The following arenon-limiting examples. The normal range of alanine transaminase is fromabout 7 to about 56 units per liter of serum. The normal range ofaspartate transaminase is from about 5 to about 40 units per liter ofserum. Bilirubin is measured using standard assays. Normal bilirubinlevels are usually less than about 1.2 mg/dL. Serum albumin levels aremeasured using standard assays. Normal levels of serum albumin are inthe range of from about 35 to about 55 g/L. Prolongation of prothrombintime is measured using standard assays. Normal prothrombin time is lessthan about 4 seconds longer than control.

A therapeutically effective amount of antiviral agent is one that iseffective to increase liver function by at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, or more. Forexample, a therapeutically effective amount of antiviral agent is anamount effective to reduce an elevated level of a serum marker of liverfunction by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more, or to reduce the level of theserum marker of liver function to within a normal range. Atherapeutically effective amount of IFN-γ is also an amount effective toincrease a reduced level of a serum marker of liver function by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or more, or to increase the level of the serum marker ofliver function to within a normal range.

Method of Reducing Risk of Hepatic Cancer

The present invention provides methods of reducing the risk that anindividual will develop hepatic cancer. The methods involveadministering an antiviral agent, as describe above, wherein viral loadis reduced in the individual, and wherein the risk that the individualwill develop hepatic cancer is reduced. An effective amount of antiviralagent is one that reduces the risk of hepatic cancer by at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, or more. Whether the risk of hepatic cancer is reducedcan be determined in, e.g., study groups, where individuals treatedaccording to the methods of the invention have reduced incidence ofhepatic cancer.

Subjects Suitable for Treatment

Individuals who have been clinically diagnosed as infected with ahepatitis virus, particularly HCV, are suitable for treatment with themethods of the instant invention. Individuals who are infected with HCVare identified as having HCV RNA in their blood, and/or having anti-HCVantibody in their serum. Such individuals include naive individuals(e.g., individuals not previously treated for HCV) and individuals whohave failed prior treatment for HCV (“treatment failure” patients).Treatment failure patients include non-responders (e.g., individuals inwhom the HCV titer was not significantly or sufficiently reduced by aprevious treatment for HCV); and relapsers (e.g., individuals who werepreviously treated for HCV, whose HCV titer decreased, and subsequentlyincreased). In particular embodiments of interest, individuals have anHCV titer of at least about 10⁵, at least about 5×10⁵, or at least about10⁶, genome copies of HCV per milliliter of serum.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1

An individual presenting with an HCV infection is treated with IFN-α. Atypical patient presents with about 10⁵ to 10⁷ genome copies of HCV permilliliter serum. IFN-α is administered in a drug delivery system thatincludes IFN-α in at a concentration of the amounts of 63-189 μg forrelease over one week or 126-378 μg for release over two week timeperiods.

In one series of treatment regimens, IFN-α is administered using asubcutaneous pump to achieve zero order input levels at 40 μg/dayinfusion of the drug subcutaneously.

The concentration of IFN-α in the serum, as well as the viral titer, aremeasured at various time points, e.g., 0 hour, 6 hours, 12 hours, 24hours, 48 hours, 4 days, 7 days, 15 days. The results are shown in FIGS.6. Similar measurements are continued for a period of six months everymonth after therapy is discontinued.

Example 2

IFN-α is administered in a range of from 200 mg to 500 mg in a volume offrom about 0.2 to 0.5 ml by subcutaneous injection.

Typical Drug Loadings are as follows: A Drug loading of 0.1% w/wprovides for a “burst” or loading dose of 10-50%. Thus, 0.1% (0.1 g/100g) of a 200 mg dose is 200 μg and 5-50% of that released dose in 1248hours is 10 μg-100 μg (first order release), with the balance of thedose released in a zero-order fashion over the course of 10-16 days(e.g. −5-10 μg/day).

In another dosing regimen, the drug loading is adjusted and the“burst-controlled” to provide adjusted release profiles: Drug loadingsof 0.5% (0.5 g/100 g=0.005) of a 200 mg dose would provide for 1 mgdoses and therefore release profiles of as much as 1-month withappropriate control of burst (5-20%) and daily maintenance releaseprofiles of as much as 20 μg/day as needed in a zero order fashion.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method for treating hepatitis C virus infection in an individual,the method comprising: administering a composition comprisinginterferon-α (IFN-α) in an amount effective to achieve a first serumconcentration of IFN-α that is at least about 80% of the maximumtolerated dose (MTD) within a first period of time of about 24 to 48hours, followed by a second concentration of IFN-α that is about 50% orless than the MTD, which second concentration is maintained for a secondperiod of time of at least seven days.
 2. The method of claim 1, whereina sustained viral response is achieved.
 3. The method of claim 1,further comprising administering IFN-γ for a period of from about 1 dayto about 14 days before administration of IFN-α.
 4. The method of claim1, wherein IFN-α is administered in a depot.
 5. The method of claim 1,wherein IFN-α is administered by continuous infusion.
 6. The method ofclaim 5, wherein said continuous infusion administration is achievedwith a pump.
 7. The method of claim 1, wherein IFN-α is administered bya single subcutaneous injection followed by continuous infusion using apump.
 8. A method of treating hepatitis C virus infection in anindividual, the method comprising: administering IFN-α in a dosingregimen comprising a first phase and a second phase, wherein, in thefirst phase, a first serum concentration of IFN-α is achieved that is atleast about 80% of the maximum tolerated dose (MTD) within a firstperiod of time of about 24 hours, wherein in the second phase, the ratioof the highest IFN-α serum concentration to the lowest serum IFN-αconcentration, measured over any 24-hour period during the second phase,is less than 3, and wherein the highest concentration of IFN-α duringthe second phase is about 50% or less than the MTD.
 9. The method ofclaim 8, wherein the ratio of the highest IFN-α serum concentration tothe lowest serum IFN-α concentration, measured over any 24-hour periodduring the second period of time is about
 1. 10. A method for treatinghepatitis C virus infection in an individual, the method comprising:administering a composition comprising consensus interferon-α (CIFN) inan amount effective to achieve a first serum concentration of CIFN thatis at least about 80% of the maximum tolerated dose (MTD) within a firstperiod of time of about 24 hours, followed by a second concentration ofCIFN that is about 50% or less than the MTD, which second concentrationis maintained for a second period of time of at least seven days.
 11. Amethod of treating hepatitis C virus infection in an individual, themethod comprising: administering consensus IFN-α (CIFN) in a dosingregimen comprising a first phase and a second phase, wherein, in thefirst phase, a first serum concentration of CIFN is achieved that is atleast about 80% of the maximum tolerated dose (MTD) within a firstperiod of time of about 24 hours, wherein in the second phase, the ratioof the highest CIFN serum concentration to the lowest serum CIFNconcentration, measured over any 24-hour period during the second phase,is less than 3, and wherein the highest concentration of CIFN during thesecond phase is about 50% or less than the MTD.
 12. A method of treatinghepatitis C virus infection in an individual, the method comprising:administering IFN-α in a dosing regimen comprising a first phase and asecond phase, wherein, in the first phase, a first serum concentrationC1max of IFN-α is achieved within a first period of time of about 24hours, wherein in the second phase, a Csus is achieved that is about 50%of C1max or less, and wherein the area under the curve, defined by IFN-αserum concentration as a function of time, during any 24-hour timeperiod in the second phase is no greater than the area under the curveof day 2 to day 3 as shown in FIG.
 2. 13. A method of treating hepatitisC virus infection in an individual, the method comprising: administeringconsensus IFN-α (CIFN) in a dosing regimen comprising a first phase anda second phase, wherein, in the first phase, a first serum concentrationC1max of CIFN is achieved within a first period of time of about 24hours, wherein in the second phase, a Csus is achieved that is about 50%of C1max or less, and wherein the area under the curve, defined by CIFNserum concentration as a function of time, during any 24-hour timeperiod in the second phase is no greater than the area under the curveof day 2 to day 3 as shown in FIG. 2.